Biotechnol. Prog. 1990, 6, 198-202
198
Genetic Engineering of Structural Protein Polymers Joseph Cappello,’ John Crissman, Mary Dorman, Marcia Mikolajczak, Garret Textor, Magda Marquet, and Franco Ferrari Protein Polymer Technologies, Inc., 10655 Sorrento Valley Road, San Diego, California 92121
Genetic and protein engineering are components of a new polymer chemistry that provide the tools for producing macromolecular polyamide copolymers of diversity and precision far beyond the current capabilities of synthetic polymer chemistry. The genetic machinery allows molecular control of chemical and physical chain properties. Nature utilizes this control to formulate protein polymers into materials with extraordinary mechanical properties, such as the strength and toughness of silk and the elasticity and resilience of mammalian elastin. The properties of these materials have been attributed to the presence of short repeating oligopeptide sequences contained in the proteins, fibroin, and elastin. We have produced homoblock protein polymers consisting exclusively of silk-like crystalline blocks and elastin-like flexible blocks. We have demonstrated that each homoblock polymer as produced by microbial fermentation exhibits measurable properties of crystallinity and elasticity. Additionally, we have produced alternating block copolymers of various amounts of silk-like and elastin-like blocks, ranging from a ratio of 1:4 to 2:1, respectively. The crystallinity of each copolymer varies with the amount of crystalline block interruptions. The production of fiber materials with custom-engineered mechanical properties is a potential outcome of this technology.
Introduction The biological genetic system synthesizes protein chains with absolute control over molecular weight, composition, sequence, and stereochemistry. Genetic engineering and biotechnology provide the tools to take advantage of this system for the production of engineered structural proteins. In nature, structural materials are assembled from several classes of biological macromolecules, polysaccharides, and proteins. Clearly, the more versatile and functional class is the proteins. The examination of natural structural proteins has shown that many consist of short repetitive sequences of amino acids. These oligopeptide sequences encode a basic structural property that allows specific interaction to occur between similar oligopeptides. The repetition of the amino acid sequence propagates that structural property over a distance as measured linearly on the extended chain. The assembly of similar structural chains creates a macromolecular polymeric material, the physical and mechanical properties of which are determined by the repeating amino acid sequence. In most natural structural proteins, the oligopeptide repeats are not perfect. Amino acid substitutions often exist throughout the protein, due presumably to nature’s engineering of segmental functionality into the chains and also to an attempt to minimize the repetitiveness of the genetic template encoding these proteins. We have used the tools of biotechnology to design and produce new proteins consisting of perfect repeating oligopeptide blocks. We have defined a protein polymer as a protein consisting primarily of exact tandemly repeated blocks of amino acid sequence, which we term sequence monomers. Protein polymer technology is the design of sequence monomers and the assembly of these monomers singly into homopolymers or in combinations into block copoly8756-7938/90/3006-0198$02.50/0
mers. Only by designing and characterizing polymers consisting exclusively of identical sequence blocks can the physical uniformity of its structure be evaluated. The structural effect of changes in the block’s amino acid sequence can also be assessed. The success of engineered protein polymers in material applications will depend on the expertise with which one can predictably create materials with specified physical and chemical properties.
Methods and Results Figure 1displays the amino acid sequences of four examples of SLP (silk-like protein) and SELP (silk-elastinlike protein) sequence monomers differing both in the composition and lengths of the monomer repeats and the periodicity of alternating repeats. The methods for their synthesis, cloning, and characterization have been previously presented (Ferrari et al., 1987). The polymerization of these monomers into genes encoding high molecular weight protein polymers and the expression of their products in E. coli have also been described and are outlined here in general terms (Figure 2A,B). The production of these products and their purification will be described in detail elsewhere (manuscript in preparation). For the purposes of the characterizations described here, 0.5-1.5 g of each polymer was purified from other E . coli cellular components by extraction with concentrated LiBr solution and extensive dialysis against water, followed by lyophilization. Protein concentration prior to lyophilization was 3-10 mg/mL. Protein purities were determined by amino acid compositional analysis to be greater than 90% (data not shown). The samples obtained after lyphilization varied in their dry powder morphologies from crystalline paperlike flakes for SLP3 and SLP4, to light,
0 1990 American Chemical Society and American
Institute of Chemical Engineers
199
Blotechnol. h g . , 1990. Vol. 6, No. 3 A
I Wannmcr qcnr
3
t iqstien
[(wMGS)9uU;v]
-
SLP3 HOWOMEP j I f L P 3 I 1 9 - S L P 3 PDLYMCP ( 1 178 AMlMO ACIDS M W - 8 3 K )
c) A r c c p t r r Plasmid
C o n f l r n correct monomer gcnr niiclrotide scqucoec
--
= OWE SLP UNIT (GAWCS) 4ODIlIOWIL AHIWO K I D S
4
w
Into Ilectcria
-
P u r i f y plasmid DNA f r o m indi vidual clanr,
- O N E CLP UNIT (YPCYC) - O W E TYPOSINC PESlOUC (Y)
Figure 1. SLP and SELP sequence monomer blocks.
fluffy puffs for SELP1, to a fibrous spongelike mat for SELP3 (Figure 3). Differences in their solubilities were also quite evident from their behavior during dialysis. Upon reduction of the LiRr concentration, SLP3 and -4 quantitatively precipitated from solution, forming a white particulate suspension. SELPl and SELP3 were dialyzed completely against water a t 4 "C with no precipitation. After several days under these conditions, SELPl formed a semiopaque gel. Gel formation was apparent with SELP3 only after longer time periods. Characterization of the 0 Crystalline Protein Polymer Class. Examples of the SLP, silk-like protein, polymer class were designed to adopt a crystalline p sheet structure typical of silk fibroin. The SLP4 polymer is comprised of approximately 160 repetitions of the six amino acid sequence glycine-alanine-glycine-alanine-glvcineserine (GAGAGS). This repeating hexamer is prevalent in silk fibroin (Lucas et al., 1957). Synthetic peptides containing this sequence have been analyzed in their crystalline states (Lucas et al., 1958). X-ray diffraction studies of poly(alanylg1ycine) have provided model unit cell crystal dimensions for @ sheet structure (Fraser et al., 1965). With use of this data for comparison, it would be possible to ascertain the physical similarity between SLP4 and shorter @ sheet forming peptides. We wanted also to test our ability to rapidly modify the properties of this homoblock polymer by disrupting the overall crystallinity by periodically inserting potentially noncrystallizing blocks. These blocks were either the pentapeptide sequence from silk fibroin, glycine-alanine-alanine-glycine-tyrosine (GAAGY) (Lucas et al., 19,58),present in SLP3, or repeats of the pentapeptide sequence of mammalian elastin, valine-proline-glycine-valine-glycine (VPGVG) (Sandberg et al., 1981;Urry et al., 1976),present in the SELP's (Figure 1). X-ray diffraction diagrams of the four protein polymers, SLP3 and -4 and SELP1 and -3, as unoriented lyophilized powders qualitatively indicated the presence of crystallinity (Figure 4). The crystalline arrangement of chains in all four polymers is similar, as indicated by the similarity in position and relative intensity of the diffraction rings. Since all four contain the same GAGAGS basic crystalline repeat sequence, we would expect the crystallized blocks of each polymer to be similar in structure. Of the four polymers, SLP3 and SELP3 gave the most diffuse scattering rings, indicating a relatively lower degree of crystallinity than that of SLP4 and SELPI. This is consistent with the fact that SELP3 contains the highest composition of presumably non-fl sheet ELP (elastin-
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Produce ) . l i a e r f r o m individual
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0
-
Choose dcslrcd w l c c r l a r r c l q h t
Figure 2. (A) Genetic engineering of gene monomer sequence. (€3) Genetic engineering of polymer gene.
like) blocks. Why SLP3 would be less crystalline than SELPl cannot a t this time be explained. The determination of unit cell dimensions and the assignment of geometry are in progress. Manually stroked polymer films were cast from formic acid onto silver chloride glass slides and studied by FTIR dichroism, according to the methods of Fraser et al. (1965). The profiles obtained for all four polvmers indicated the presence of mixtures of fi and random structure (Figure 5A-D). The absorbances a t approximately 1650 and 1630 cm-', the amide I region, indicated the relative content of random and hydrogen-bonded 0structure, respectively, in each sample (see Fraser et al., 1965, for peak assignments). SLP4 and SELPl had greater 0 structure than did SLP3 and SELP3. Although the degree of orientation in these stroked films can depend on the experimental conditions, the degree of chain alignment achieved here as judged by the dichroic ratio of each peak was greatest for SLP3, SLP4, and SELP3. The ahsor-
Biofechnol. Rog., 1990, Val. 6,
200
A SLP3 POWDER
No. 3
B SLP4POWDER
-,,&- 3., .< '*a.
C SELPl POWDER
D SELP3 POWDER
i
Figure 3. SLP and SELP polymer unoriented lyophilized powders.
bances of the amide I and I1 regions (approximately 1630 and 1530 cm-l, respectively) in SLP3 and SLP4 were greater when polarized perpendicular to the stroking direction; therefore, chains were aligned in the direction of stroking. In SELP3, the stroking created a chain alignment that gave increased absorption when polarized parallel to the stroking direction. The alignment of SELP3 is consistent with a cross-@conformation where the alignment of chains is perpendicular to the stroking direction, similar to the results obtained for synthetic poly(alanylglycine) (Fraser et al., 1965). The alignment of SLP3 and -4 is similar to the observed alignment of chains in natural silk fibers (Fraser et al., 1965). We do not yet know the extent to which variation in experimental conditions affects chain alignment in manually stroked films. Investigation of this phenomenon is in progress.
Summary and Discussion
Our goal is to correlate the effects of compositional changes on the material properties of protein-based mate-
rials. We have developed genetic expression and protein purification methods for the efficient production of synthetically designed protein polymers. These methods allow us precise control over compositional variance. The evaluation of some of the properties affected by the inclusion of structurally dissimilar amino acid blocks in a specific linear sequence along the protein chain is presented . We have demonstrated that examples of our silk-like polymer class produce organized crystalline structuressimilar to the p sheet structures found in natural silk. We have demonstrated that we can effect the degree of crystallinity by periodically disrupting the crystalline blocks with blocks of amino acids, which cannot be accommodated within the crystal packing dimensions observed for glycylalanine sequential polypeptides. The inclusion of the pentapeptide sequence, GAAGY, containing the bulky tryrosine side chain, in SLP3 decreases the crystallinity of the poly(GAGAGS) blocks present in SLP4, as determined by increased diffusivity of X-ray powder diffrac-
201
Biotechnol. Rug..,1990, Vol. 6, No. 3
A SLP3 POWDER
6 SLP4POWDER
.
C SELPl POWDER
..
D SELP3 POWDER
Figure 4. X-ray diffraction of protein polymer lyophilized powders.
tion rings. It is interesting to note that this decrease in crystallinity in the lyophilized powders of SLP3 versus SLP4 cannot be observed visually. Both powders appear as paperlike flakes (Figure 3). The periodic insertion of non-0 sheet, flexible sequence segments such as those modeled after the repeating oligopeptides of mammalian elastin (Urry et al., 1976) was were designed to decrease the overall crystallinity of the chains and potentially impart some flexibility to the structure. The presence of the elastin-like blocks as well as changes in their length or position within the SELP monomers influence the molecular chain properties, as evident from the changes in solubility of the polymers in water. SLP3 and SLP4 are completely insoluble in aqueous solutions. The disruption of the crystalline silk-like blocks with either 4 or 8 units of ELP blocks in SELPl and SELP3, respectively, renders these polymers water soluble. After several days in solution, SELYl and, later, SELP3 form gels. This increase in water solubility is accomplished with little change in the chemical proper-
ties of the SELP protein chain. If anything, the overall hydrophobicity of the SELP copolymers increases due to their increased composition of valine. This is consistent with the hypothesis that the ELP blocks contribute to the chain properties of the SELP copolymer by disrupting the crystallization of the silk-like structure. Upon drying, none of the polymers can be redissolved in water. Presumably, once the polymers are induced to crystallize, the cohesion of the silk-like blocks, even when interrupted, dominates the solubility properties. The difference in crystallinity between the SELP's and the SLP's is easily observed in the morphological properties of the dried powders (Figure 3). The three-dimensional bulk of the SELP powders indicates some degree of chain entanglement and network formation.
Acknowledgment We acknowledge the technical assistance provided by Drs. David Tirrell, Harold Modler, and Kevin McGrath
Biotechnol. Prog., 1990,Vol. 6, No. 3
202
LL-
lloo
zmo
zino
ism
15uo
L I!OF
I
00
1JW
1100
2100
2300
1900
I100
IlW
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Figure 5. FTIR dichroism of SLP and SELP stroked films.
on the physical characterization of the samples. We thank them for their creative input and their continued support.
Literature Cited Ferrari, F. A.; Richardson, C.; Chambers, J.; Causey, S. C.; Pollock, T. J. Construction of synthetic DNA and its use in large polypeptide synthesis. Patent application 1987, international publication number W088/03533. Fraser, R. D. B.; MacRae, T. P.; Stewart, F. H. C.; Suzuki, E. J. Mol. Biol. 1965, 11, 706. Lucas, F.; Shaw, J. T. B.; Smith, S. G. The amino acid sequence
in a fraction of the fibroin of Bombyx mori. Biochem. J . 1957, 66, 468-479. Lucas, F.; Shaw, J. T. B.; Smith, S. G. The silk fibroins. Ado. Protein Chem. 1958, 13, 107-242. Sandberg, L. B.; Soskel, N. T.; Leslie, J. B. Elastin structure, biosynthesis, and relation to disease states. N . Engl. J. Med. 1981,304,566-579. Urry, D. W.; Okamoto, K.; Harris, R. D.; Hendrix, C. F.; Long, M. M. Synthetic, cross-linked polypentapeptide of tropoelastin: An anisotropic, fibrillar elastomer. Biochemistry 1976, 25,4083-4089. Accepted May 18, 1990.
198
Genetic Engineering of Structural Protein Polymers Joseph Cappello,’ John Crissman, Mary Dorman, Marcia Mikolajczak, Garret Textor, Magda Marquet, and Franco Ferrari Protein Polymer Technologies, Inc., 10655 Sorrento Valley Road, San Diego, California 92121
Genetic and protein engineering are components of a new polymer chemistry that provide the tools for producing macromolecular polyamide copolymers of diversity and precision far beyond the current capabilities of synthetic polymer chemistry. The genetic machinery allows molecular control of chemical and physical chain properties. Nature utilizes this control to formulate protein polymers into materials with extraordinary mechanical properties, such as the strength and toughness of silk and the elasticity and resilience of mammalian elastin. The properties of these materials have been attributed to the presence of short repeating oligopeptide sequences contained in the proteins, fibroin, and elastin. We have produced homoblock protein polymers consisting exclusively of silk-like crystalline blocks and elastin-like flexible blocks. We have demonstrated that each homoblock polymer as produced by microbial fermentation exhibits measurable properties of crystallinity and elasticity. Additionally, we have produced alternating block copolymers of various amounts of silk-like and elastin-like blocks, ranging from a ratio of 1:4 to 2:1, respectively. The crystallinity of each copolymer varies with the amount of crystalline block interruptions. The production of fiber materials with custom-engineered mechanical properties is a potential outcome of this technology.
Introduction The biological genetic system synthesizes protein chains with absolute control over molecular weight, composition, sequence, and stereochemistry. Genetic engineering and biotechnology provide the tools to take advantage of this system for the production of engineered structural proteins. In nature, structural materials are assembled from several classes of biological macromolecules, polysaccharides, and proteins. Clearly, the more versatile and functional class is the proteins. The examination of natural structural proteins has shown that many consist of short repetitive sequences of amino acids. These oligopeptide sequences encode a basic structural property that allows specific interaction to occur between similar oligopeptides. The repetition of the amino acid sequence propagates that structural property over a distance as measured linearly on the extended chain. The assembly of similar structural chains creates a macromolecular polymeric material, the physical and mechanical properties of which are determined by the repeating amino acid sequence. In most natural structural proteins, the oligopeptide repeats are not perfect. Amino acid substitutions often exist throughout the protein, due presumably to nature’s engineering of segmental functionality into the chains and also to an attempt to minimize the repetitiveness of the genetic template encoding these proteins. We have used the tools of biotechnology to design and produce new proteins consisting of perfect repeating oligopeptide blocks. We have defined a protein polymer as a protein consisting primarily of exact tandemly repeated blocks of amino acid sequence, which we term sequence monomers. Protein polymer technology is the design of sequence monomers and the assembly of these monomers singly into homopolymers or in combinations into block copoly8756-7938/90/3006-0198$02.50/0
mers. Only by designing and characterizing polymers consisting exclusively of identical sequence blocks can the physical uniformity of its structure be evaluated. The structural effect of changes in the block’s amino acid sequence can also be assessed. The success of engineered protein polymers in material applications will depend on the expertise with which one can predictably create materials with specified physical and chemical properties.
Methods and Results Figure 1displays the amino acid sequences of four examples of SLP (silk-like protein) and SELP (silk-elastinlike protein) sequence monomers differing both in the composition and lengths of the monomer repeats and the periodicity of alternating repeats. The methods for their synthesis, cloning, and characterization have been previously presented (Ferrari et al., 1987). The polymerization of these monomers into genes encoding high molecular weight protein polymers and the expression of their products in E. coli have also been described and are outlined here in general terms (Figure 2A,B). The production of these products and their purification will be described in detail elsewhere (manuscript in preparation). For the purposes of the characterizations described here, 0.5-1.5 g of each polymer was purified from other E . coli cellular components by extraction with concentrated LiBr solution and extensive dialysis against water, followed by lyophilization. Protein concentration prior to lyophilization was 3-10 mg/mL. Protein purities were determined by amino acid compositional analysis to be greater than 90% (data not shown). The samples obtained after lyphilization varied in their dry powder morphologies from crystalline paperlike flakes for SLP3 and SLP4, to light,
0 1990 American Chemical Society and American
Institute of Chemical Engineers
199
Blotechnol. h g . , 1990. Vol. 6, No. 3 A
I Wannmcr qcnr
3
t iqstien
[(wMGS)9uU;v]
-
SLP3 HOWOMEP j I f L P 3 I 1 9 - S L P 3 PDLYMCP ( 1 178 AMlMO ACIDS M W - 8 3 K )
c) A r c c p t r r Plasmid
C o n f l r n correct monomer gcnr niiclrotide scqucoec
--
= OWE SLP UNIT (GAWCS) 4ODIlIOWIL AHIWO K I D S
4
w
Into Ilectcria
-
P u r i f y plasmid DNA f r o m indi vidual clanr,
- O N E CLP UNIT (YPCYC) - O W E TYPOSINC PESlOUC (Y)
Figure 1. SLP and SELP sequence monomer blocks.
fluffy puffs for SELP1, to a fibrous spongelike mat for SELP3 (Figure 3). Differences in their solubilities were also quite evident from their behavior during dialysis. Upon reduction of the LiRr concentration, SLP3 and -4 quantitatively precipitated from solution, forming a white particulate suspension. SELPl and SELP3 were dialyzed completely against water a t 4 "C with no precipitation. After several days under these conditions, SELPl formed a semiopaque gel. Gel formation was apparent with SELP3 only after longer time periods. Characterization of the 0 Crystalline Protein Polymer Class. Examples of the SLP, silk-like protein, polymer class were designed to adopt a crystalline p sheet structure typical of silk fibroin. The SLP4 polymer is comprised of approximately 160 repetitions of the six amino acid sequence glycine-alanine-glycine-alanine-glvcineserine (GAGAGS). This repeating hexamer is prevalent in silk fibroin (Lucas et al., 1957). Synthetic peptides containing this sequence have been analyzed in their crystalline states (Lucas et al., 1958). X-ray diffraction studies of poly(alanylg1ycine) have provided model unit cell crystal dimensions for @ sheet structure (Fraser et al., 1965). With use of this data for comparison, it would be possible to ascertain the physical similarity between SLP4 and shorter @ sheet forming peptides. We wanted also to test our ability to rapidly modify the properties of this homoblock polymer by disrupting the overall crystallinity by periodically inserting potentially noncrystallizing blocks. These blocks were either the pentapeptide sequence from silk fibroin, glycine-alanine-alanine-glycine-tyrosine (GAAGY) (Lucas et al., 19,58),present in SLP3, or repeats of the pentapeptide sequence of mammalian elastin, valine-proline-glycine-valine-glycine (VPGVG) (Sandberg et al., 1981;Urry et al., 1976),present in the SELP's (Figure 1). X-ray diffraction diagrams of the four protein polymers, SLP3 and -4 and SELP1 and -3, as unoriented lyophilized powders qualitatively indicated the presence of crystallinity (Figure 4). The crystalline arrangement of chains in all four polymers is similar, as indicated by the similarity in position and relative intensity of the diffraction rings. Since all four contain the same GAGAGS basic crystalline repeat sequence, we would expect the crystallized blocks of each polymer to be similar in structure. Of the four polymers, SLP3 and SELP3 gave the most diffuse scattering rings, indicating a relatively lower degree of crystallinity than that of SLP4 and SELPI. This is consistent with the fact that SELP3 contains the highest composition of presumably non-fl sheet ELP (elastin-
U B
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C hoasc production c l a w
3
I
Occtrrphbrc¶ls flriyiri
Produce ) . l i a e r f r o m individual
cloner Determine
0
-
Choose dcslrcd w l c c r l a r r c l q h t
Figure 2. (A) Genetic engineering of gene monomer sequence. (€3) Genetic engineering of polymer gene.
like) blocks. Why SLP3 would be less crystalline than SELPl cannot a t this time be explained. The determination of unit cell dimensions and the assignment of geometry are in progress. Manually stroked polymer films were cast from formic acid onto silver chloride glass slides and studied by FTIR dichroism, according to the methods of Fraser et al. (1965). The profiles obtained for all four polvmers indicated the presence of mixtures of fi and random structure (Figure 5A-D). The absorbances a t approximately 1650 and 1630 cm-', the amide I region, indicated the relative content of random and hydrogen-bonded 0structure, respectively, in each sample (see Fraser et al., 1965, for peak assignments). SLP4 and SELPl had greater 0 structure than did SLP3 and SELP3. Although the degree of orientation in these stroked films can depend on the experimental conditions, the degree of chain alignment achieved here as judged by the dichroic ratio of each peak was greatest for SLP3, SLP4, and SELP3. The ahsor-
Biofechnol. Rog., 1990, Val. 6,
200
A SLP3 POWDER
No. 3
B SLP4POWDER
-,,&- 3., .< '*a.
C SELPl POWDER
D SELP3 POWDER
i
Figure 3. SLP and SELP polymer unoriented lyophilized powders.
bances of the amide I and I1 regions (approximately 1630 and 1530 cm-l, respectively) in SLP3 and SLP4 were greater when polarized perpendicular to the stroking direction; therefore, chains were aligned in the direction of stroking. In SELP3, the stroking created a chain alignment that gave increased absorption when polarized parallel to the stroking direction. The alignment of SELP3 is consistent with a cross-@conformation where the alignment of chains is perpendicular to the stroking direction, similar to the results obtained for synthetic poly(alanylglycine) (Fraser et al., 1965). The alignment of SLP3 and -4 is similar to the observed alignment of chains in natural silk fibers (Fraser et al., 1965). We do not yet know the extent to which variation in experimental conditions affects chain alignment in manually stroked films. Investigation of this phenomenon is in progress.
Summary and Discussion
Our goal is to correlate the effects of compositional changes on the material properties of protein-based mate-
rials. We have developed genetic expression and protein purification methods for the efficient production of synthetically designed protein polymers. These methods allow us precise control over compositional variance. The evaluation of some of the properties affected by the inclusion of structurally dissimilar amino acid blocks in a specific linear sequence along the protein chain is presented . We have demonstrated that examples of our silk-like polymer class produce organized crystalline structuressimilar to the p sheet structures found in natural silk. We have demonstrated that we can effect the degree of crystallinity by periodically disrupting the crystalline blocks with blocks of amino acids, which cannot be accommodated within the crystal packing dimensions observed for glycylalanine sequential polypeptides. The inclusion of the pentapeptide sequence, GAAGY, containing the bulky tryrosine side chain, in SLP3 decreases the crystallinity of the poly(GAGAGS) blocks present in SLP4, as determined by increased diffusivity of X-ray powder diffrac-
201
Biotechnol. Rug..,1990, Vol. 6, No. 3
A SLP3 POWDER
6 SLP4POWDER
.
C SELPl POWDER
..
D SELP3 POWDER
Figure 4. X-ray diffraction of protein polymer lyophilized powders.
tion rings. It is interesting to note that this decrease in crystallinity in the lyophilized powders of SLP3 versus SLP4 cannot be observed visually. Both powders appear as paperlike flakes (Figure 3). The periodic insertion of non-0 sheet, flexible sequence segments such as those modeled after the repeating oligopeptides of mammalian elastin (Urry et al., 1976) was were designed to decrease the overall crystallinity of the chains and potentially impart some flexibility to the structure. The presence of the elastin-like blocks as well as changes in their length or position within the SELP monomers influence the molecular chain properties, as evident from the changes in solubility of the polymers in water. SLP3 and SLP4 are completely insoluble in aqueous solutions. The disruption of the crystalline silk-like blocks with either 4 or 8 units of ELP blocks in SELPl and SELP3, respectively, renders these polymers water soluble. After several days in solution, SELYl and, later, SELP3 form gels. This increase in water solubility is accomplished with little change in the chemical proper-
ties of the SELP protein chain. If anything, the overall hydrophobicity of the SELP copolymers increases due to their increased composition of valine. This is consistent with the hypothesis that the ELP blocks contribute to the chain properties of the SELP copolymer by disrupting the crystallization of the silk-like structure. Upon drying, none of the polymers can be redissolved in water. Presumably, once the polymers are induced to crystallize, the cohesion of the silk-like blocks, even when interrupted, dominates the solubility properties. The difference in crystallinity between the SELP's and the SLP's is easily observed in the morphological properties of the dried powders (Figure 3). The three-dimensional bulk of the SELP powders indicates some degree of chain entanglement and network formation.
Acknowledgment We acknowledge the technical assistance provided by Drs. David Tirrell, Harold Modler, and Kevin McGrath
Biotechnol. Prog., 1990,Vol. 6, No. 3
202
LL-
lloo
zmo
zino
ism
15uo
L I!OF
I
00
1JW
1100
2100
2300
1900
I100
IlW
iOC
Figure 5. FTIR dichroism of SLP and SELP stroked films.
on the physical characterization of the samples. We thank them for their creative input and their continued support.
Literature Cited Ferrari, F. A.; Richardson, C.; Chambers, J.; Causey, S. C.; Pollock, T. J. Construction of synthetic DNA and its use in large polypeptide synthesis. Patent application 1987, international publication number W088/03533. Fraser, R. D. B.; MacRae, T. P.; Stewart, F. H. C.; Suzuki, E. J. Mol. Biol. 1965, 11, 706. Lucas, F.; Shaw, J. T. B.; Smith, S. G. The amino acid sequence
in a fraction of the fibroin of Bombyx mori. Biochem. J . 1957, 66, 468-479. Lucas, F.; Shaw, J. T. B.; Smith, S. G. The silk fibroins. Ado. Protein Chem. 1958, 13, 107-242. Sandberg, L. B.; Soskel, N. T.; Leslie, J. B. Elastin structure, biosynthesis, and relation to disease states. N . Engl. J. Med. 1981,304,566-579. Urry, D. W.; Okamoto, K.; Harris, R. D.; Hendrix, C. F.; Long, M. M. Synthetic, cross-linked polypentapeptide of tropoelastin: An anisotropic, fibrillar elastomer. Biochemistry 1976, 25,4083-4089. Accepted May 18, 1990.
